Fluorescence emission properties of 8-azaxanthine and its N-alkyl derivatives: Excited-state proton transfer, and potential applications in enzymology

We have investigated the emission properties of 8-azaxanthine (8-azaXan) and its triazolo N-alkyl derivatives, with particular emphasis on N(8)-methyl-8-azaXan. In aqueous medium, both 8-azaXan and its 8-alkyl congener emit at 420 nm, with an unusually large Stokes’ shift, and decay times of 9 and 12 ns, respectively. With the parent 8-azaXan, emission is observed over a narrow pH range (2–6), where it is partially or predominantly in its neutral form in the ground state (pKa 4.9), whereas for 8-methyl-8-azaXan emission is observed in the pH range 2–12, hence for both the neutral and monoanionic species (pKa 7.2). Fluorescence excitation spectra are in accord with the absorption spectra at all pH values. In anhydrous methanol, 8-azaX, in its neutral form, emits at about 335 nm. In 10% aqueous methanol, acidified to pH ∼3, two emission bands are observed, at 420 and 335 nm. With the 8-methyl derivative, dual emission at 420 and ∼340 nm is observed in anhydrous methanol, but in isopropanol and acetonitrile the 420 band virtually disappears. It is concluded that the 420 nm emission band, ascribed to the monoanionic form of 8-methyl-8-azaXan, appears in neutral or acidic media as the result of rapid deprotonation of the N(3)–H in the excited states of both 8-azaXan and its 8-alkyl derivative. This leads to phototautomerism in the 8-azaXan monoanion which, in the ground state, is present in the form resulting from dissociation of the triazole proton, i.e., N(8)–H. This proton transfer is slowed down in non-aqueous solvents, where emission from the neutral molecules (335–340 nm) is observed. The foregoing interpretation is supported by determination, with the aid of the Foerster cycle, of the excited state pK* values, close to −0.5 for 8-methyl-8-azaXan. The strong emission of 8-azaXan and its N-alkyl derivatives should prove useful in studies on enzyme/ligand interactions, several examples of which are proposed. In particular, we report that 8-azaXan is a substrate for purine nucleoside phosphorylase II from E. coli, and show that the reaction can be monitored fluorimetrically.